Combinations of fermentation techniques and various carbon energy substrates, and particular microorganism strains can produce valuable intracellular enzymes. It is highly desirable to break the cells and obtain the proteins contained therein, particularly the valuable enzymes, without destroying the enzymes.
A variety of approaches have been employed for the disruption of yeat cells in order to release the contained proteins, particularly enzymes. Mechanical disruption techniques have included ball milling, grinding, sonication, and the like, usually with cooling to minimize protein breakdown. Mechanical disruptions, however, generally result in some degradation of the enzymes and the resultant extremely heterogeneous mixture of cell fragments can prove difficult to separate effectively from the soluble fraction. Added enzymes have been employed to effect a destruction or disruption of the cell walls and release of the cellular contents. However, added enzymes can be expensive and introduce foreign proteins and may pose undesired separatory problems, or cause desired side effects in uses of the desired enzymes.
Chemical methods have been employed to cause autolysis (plasmolysis), including various hydrocarbons such as toluene; carbonyl compounds including ethyl acetate, acetone, other dialkyl ketones; and other chemicals such as ethyl ether and alcohols such as C.sub.1 to C.sub.4 alkanols. These chemical approaches have had varying results depending upon the organism, its growth conditions, and the time of exposure to and concentration of the solvent. Some have been hazardous since at desired treatment temperatures volatility may produce flammable, indeed, explosive conditions. Some require high concentrations of lytic solvents which may lead to separation problems in subsequent purification steps.
Needed, still, are new treatments to produce good results in protein release from cells, yet employing safe materials, mild conditions and a minimum of added lytic component.